Nuclear Magnetic Resonance (NMR) is a resonance method for structural analysis using atomic nuclei. This method observes resonance spectra of individual atomic nuclei, so that even atoms having similar electron cloud sizes can be distinctly differentiated from each other. Studies of structural analysis by NMR using 1H, 13C and 15N are under way.
Biological components, especially amino acids are important components capable of forming proteins, and analyses of the high-order structures of many proteins using NMR are being reported as well as elucidation of their biological functions. Oxygen atoms are very important in biological components such as amino acids and sugars, but 17O NMR spectroscopy has not been sufficiently developed. One reason for this is that the natural abundance of the isotope 17O is as low as 0.038%. See Table 1 summarizing the properties of atomic nuclei of hydrogen, carbon, nitrogen and oxygen that can be used in NMR.
TABLE 1IsotopicabundanceNMR resonant(atomicfrequencyElementNuclidepercentage)a)Spin(MHz)b)NoteHydrogen 1H99.985½100.000 2H0.015115.351 3H—½106.663Radioisotope(half life12.26 years)Carbon12C98.900—13C1.10½25.144Nitrogen14N99.63417.22415N0.366½10.133Oxygen16O99.7620—17O0.038 5/213.55718O0.2000—a)IUPAC Inorganic Chemistry Division, CAWIA SIAM: Isotopic Compositions of the elements 1989. Pure Appl. Chem., 63, 991(1991); Chemistry and Industry, Vol. 48, April (1995).b)Resonant frequency in a static magnetic field of 2.35 tesla.
As shown in Table 1, the natural abundance of 17O is also significantly low as compared with 13C (1.10%) and 15N (0.366%). Thus, test compounds to be analyzed by 17O NMR must be artificially enriched in 17O by labeling oxygen atoms in the test compounds with 17O.
Carboxyl-containing compounds such as amino acids can be enriched in 17O by reacting the amino acids with H217O to replace an oxygen atom in the carboxyl group by 17O. However, such exchange reaction hardly proceeds spontaneously, so that it must be forced to proceed by using a catalyst or the like. Prior to the present invention, the reaction was performed under strongly acidic conditions by saturating H217O with hydrogen chloride gas (Non-Patent Publication: No. 4), or the carboxyl group was preliminarily subjected to alkyl esterification and then alkaline hydrolysis using sodium hydroxide (Non-Patent Publications: Nos. 2, 3, 5), etc. However, these conventional conditions involved the following problems: the exchange reaction of the carboxyl group under acidic conditions invited degradation of tryptophan, cysteine, asparagine and glutamine, and alkaline hydrolysis of alkyl ester derivatives inevitably caused racemization of optically active amino acids.
Thus, there have been demands for a method for labeling carboxyl-containing compounds with oxygen isotopes such as oxygen-17 (17O) under milder conditions.